1
Q
What does recognition of DNA seqs by DNA binding proteins allow?
A
- targeting of RNA pol to specific genes
2
Q
What do genes transcribed at high rate show?
A
- high density of RNA pol along length of gene
3
Q
Where is DNA of euk cells packaged?
A
- chromatin
4
Q
What is the Barr body?
A
- eg. of densely packaged chromatin
5
Q
Why must certain regions of chromatin be unravelled?
A
- in order for specific genes to be transcribed
- for TFs to gain access to DNA
6
Q
How are genes switched on/off through chromatin?
A
- localised unfolding and repackaging of DNA into chromatin
7
Q
Do diverse types of somatic cells have same DNA content and gene expression?
A
- same DNA content
- differential patterns of gene expression
8
Q
What do cells switch genes on/off in response to?
A
- extracellular stimuli
- eg. nutrients or signalling molecules from other cells
9
Q
What recognises signalling molecules?
A
- receptor molecules on cell surface or localised w/in cell
10
Q
Why are signalling pathways that stimulate cell growth and division usually activated for limited periods?
A
- constitutive activation can cause uncontrolled growth and cancer
11
Q
At what level are gene expression levels in euk cells mainly reg?
A
- transcriptional level
12
Q
How is coding pot of euk genomes increased?
A
- through alt pre-mRNA splicing patterns that prod distinct protein products
13
Q
What can errors in splicing patterns cause?
A
- genetic diseases
- eg. muscular atrophy
14
Q
When can gene expression also be reg?
A
- post transcriptionally at level of alt splicing, mRNA translation and/or mRNA stability
15
Q
Why are some transcripts localised to specific regions of cell?
A
- ensure targeted expression of protein
16
Q
What is the role of ribonucleases?
A
- provide activities central to process functional RNAs from 1° transcripts
- quality control systems that remove misassembled/incorrectly processed RNAs
- timely degradation of mRNA
17
Q
What is the GAL switch?
A
- model inducible genetic control system in budding yeast
- monosaccharide sugar galactose must be converted to glucose for it to be metabolised by euk cells
18
Q
Why is transcriptional control of euk genes important?
A
- for cellular differentiation, dev and cellular signalling
- response to env
19
Q
What does transcriptional control of euk genes involve?
A
- cis regulatory elements and DNA binding proteins
20
Q
How does dispersal of regulatory elements vary in humans and E. Coli?
A
- far more dispersed than E. Coli
- in adults = distal promoter
- in embryonic cDNAs = proximal promoter
21
Q
How does sex lethal gene determine sex in fruit flies?
A
- expressed form embryonic PE promotor only in females
- expressed from PL promoter further upstream in males and females
- PE and PL gen distinct but overlapping transcripts
22
Q
What is promoter region?
A
- where RNA pol assembled and lies immediately upstream of transcrip start site
23
Q
Where are TATA boxes found?
A
- promoters of highly inducible genes
24
Q
What is a TATA box?
A
- approx 30nts 5’ of TSS
25
What are CpG islands?
- clusters of dinucleotide CG w/in promoter region that are unmethylated
- -> seq CpG usually methylated
- -> seq CG rare as targeted by DNA methyltransferases and can mutate to TG when methylated
26
How can TSS of gene be mapped?
- viral RT enzymes can gen ssDNA from RNA template
- start DNA primers can be designed by knowing small part of seq and annealed to ssRNA
- DNA primer extended 5' to 3' by RT until reaches 5' end of RNA
- lengths of cDNA products measured by high res PAGE and mRNA 5' ends inferred
27
What is deletion analysis of promoter regions used for?
- map reg elements w/in promoter region
28
How is deletion analysis of promoter regions carried out?
- promoter region cloned upstream of coding seq of reporter gene w/ easily assayable activity (eg. lacZ) insuitable plasmid
- transfection of cells w plasmid allows reg expression of reporter gene
- short stretches of promoter region can be removed from either end and truncated promoter seqs reinserted into vector and tested for ability to stimulate transcrip in vivo
- by making cell extracts from transferred cells and assaying for level of reporter gene
29
When is linker scanning mutagenesis of promoters carried out?
- after promoter region defined by deletion analysis
30
How is linker scanning mutagenesis of promoters carried out?
- mutate short overlapping stretches of nucleotides to gen series of constructs, each containing randomised nucleotides w/in specific region of same length of DNA
- mutations that cause decrease in reporter protein activity define short seqs for transcriptional activity
- typically contain TATA box and promoter-proximal regions
31
How do distant enhancer elements stimulate transcrip?
- can be enhancer or silencer elements
- typically consist of arrays of recognition sites for nt-specific DNA binding proteins
- generally function in cell type specific manner and activate set of genes close together in chromosome
- enhancers composed of arrays of seq elements
32
How are transcrip regulatory elements organised?
- mammalian genes contain promoter-proximal elements, distal enhancers and promoter elements
- promoter-proximal elements found w/in ≈200 nts upstream of TSS
- enhancer regions can be 10s of hilobases upstream of TSS, w/in introns or up to 10s of kilobases downstream of coding seq
33
How are yeast genes organised?
- highly compact genome w/ 1 protein encoding gene per 2kb DNA
- promoter and terminator regions short
- TATA box ≈90 nts upstream of TSS
- less regulatory elements, typically reg by single upstream activating seq or upstream repression seq
34
Where is preinitiation complex (PIC) is assembled on promoters?
- PIC of general TF assembled at pol II promoters
35
How is preinitiation complex assembled on promoters?
- TATA box binding protein subunit of TFIID complex directly binds to TATA box seq element ≈30 nts upstream of TSS
- TATA box binding protein binds to minor groove and gen bend in DNA
- TFIIB complex binds to TFIID
- RNA pol II assoc w/ TFIIF --> binds TFIID/TFIIB complex
- TFIIE and TFIIH bind to gen PIC
- TFIIH has DNA helicase activity, separates 2 strands allowing primer synthesis
36
What do proks and euks both have in terms of transcrip reg?
- RNA pol and initiation factors that target pol to promoter regions
37
What do regulatory proteins target in euks and proks?
- proks = directly target pol
| - euks = impact pol indirectly via chromatin structure and mediator complex
38
What is the mediator complex (euk regulatory proteins target)?
- large complex of 30+ diff proteins conserved from yeast to man
39
How is transcrip reg in euks by mediator complex and TFs?
- mediator complex and general TFs allow basal level of transcrip by RNA pol II
- specific TFs are reg proteins that stimulate or repress basal level of transcrip from certain protein
40
What is a basal level of transcrip?
- nonstimulated, nonrepressed level of transcrip by RNA pol II
41
Why is linkage of functionally linked genes simpler in proks?
- bacteria have operons
| - genes expressed in same time in same amount
42
How is linkage of functionally linked genes by specific TFs experimentally investigated?
- expression levels of gene array measured upon transcrip repression of no. of test genes
- horizontal lines = diff groups
- vertical lines = diff conditions
- specific TFs 2-10% of euk proteome, but expression of most genes is reg
- achieved as transcrip control of functionally linked genes coord by sets of specific TFs
- -> these regions defined by transcriptional profiling and cluster analysis of global gene expression using microarray
- compare genes and see some activated and repressed in same pattern (clustered in groups), so can identify genes coordinately expressed w/o knowing function or running other experiments
43
What is combinatorial control?
- TFs function by it
| - small no. factors can be arranged in v large no. combos
44
What is the structure of TFs?
- modular
| - typically DNA binding domain and activation/repression domain, separated by flexible linker region
45
What is the role of activation domain in TFs?
- fusion of AD to other DNA binding proteins confers control of genes w/ cognate binding site
46
What assay clearly demonstrates modular structure of TFs?
- yeast 2-hybrid
47
How is yeast 2-hybrid carried out?
- Gal4 stimulates expression of genes w/ GAL upstream activating seq w/in promoter region in response to galactose
- DNA binding domain and activation domain fused separately to bait and prey proteins
- if bait and prey interact, Gal4 protein reconstituted and can promote transcrip of reporter gene containing promoter-proximal GAL upstream activating seq
- identifies proteins that interact in vivo w/ protein of interest and to map region of interaction
48
What are typical reporter genes used in yeast 2-hybrid assays?
- HIS3 = false +ves filtered out by using increased concs of comp inhibitor
- ADE2 = coloured colonies
- lacZ = coloured colonies
49
How do multiprotein complexes assemble on enhancers?
- enhancer regions consist of multiple reg elements that bind diff TFs cooperatively
- HMGI (DNA BP) binds to minor groove, causing it to bend
- enhancer complex containing multiple subunits assembled through large no. protein-protein and protein-DNA interactions
- -> multiple weak interactions between sTFs strengthened by interactions w/ DNA
50
What is the best characterised enhancer complex, and what is its structure?
- virus-inducible β-interferon complex
| - contains heterodimers that cooperatively bind to adj regulatory elements in HMGI presence
51
What is an example of a model genetic reg system?
- yeast GAL gene switch
52
How does the yeast GAL gene switch work?
- galactose --> glucose req Gal2 permease and 3 enzymes (Gal1, Gal7, Gal10)
- expression reg in response to galactose by sTF Gal4, regulator protein Gal80 and galactose sensor Gal3
53
What are gel shift assays used for?
- purify TFs that interact w/ reg element of known seq
54
How are gel shift assays carried out?
- short ds DNA molecule containing reg element incubated w/ fractionated nuclear extract
- complexes of DNA bound to protein have slower electrophoretic mobility than non bound DNA
- allowing identification of fractions containing cognate sTF
- reiterative cycles of fractionation based on differing physical properties
55
What question can in vitro assay of TF activity investigate?
- is purified DNA binding protein actually a TF
56
How is in vitro assay of TF activity carried out?
- performed in presence and absence of purified protein
| - using templates that either do or don't contain TF binding site
57
How is an in vitro (transfection) assay carried out?
- combined w/ in vitro to prove in cellular context
- plasmid bearing cloned sTF and reporter gene, w/ cognate sTF binding site, introd into cells and measure amounts of reporter mRNA/protein
- expect increase if cloned gene encodes cognate TF
- allows mutational analysis of sTF
58
What is the structure and role of ligand binding domains?
- ligand binding domains of nuclear receptor are folded into compact structures
- function as activation domains when bound to their hormone
- binding triggers conformational change facilitating interaction w/ coactivators
- eg. estrogen receptor when binds estrogen or tamoxifen
59
What are the characteristics of acidic activation domains?
- largely unstructured but fold upon binding coactivators
60
How can activator/coactivator interactions be reg?
- adenylate cyclase gen cAMP, which activates protein kinase A
- sTF CREB phosphorylated by catalytic subunit of protein kinase A
- CREB must be phosphorylated to bind coactivator, CREB BP
- CREB binds to cAMP response elements, allowing transcrip of genes reg by them
61
What can defects in transcrip repressor domains cause?
- uncontrolled growth --> cancer
62
How can transcrip repressors be identified?
- genetically or biochemically
63
What is base-specific recognition of DNA binding proteins?
- bind in seq specific manner
- base recognition occurs through interactions made in major groove of DNA
- res w/in α helices contact edges of bases
- basic residues may also interact w/ phosphate backbone
- bacteriophage repressors often dimers
- recognition helix inserted into major groove and supported in helix-turn-helix fold
64
What is the homeodomain fold assoc w/ in sTFs?
- morphogenesis
65
Where is the homeodomain fold found?
- euk TFs
| - similar to helix-turn-helix fold of bacterial repressors
66
What is the homeodomain fold made up of?
- 60AAs encoded by 180 nt long homeobox DNA seq
67
What is the structure and role of Hox genes?
- homeobox seqs highly conserved and readily identifiable w/ seq alignment tools
- key role in anterior-posterior dev
- clustered and order correlates w/ spatial and temporal expression
68
What is the most common DNA-binding fold in human cells?
- zinc-finger proteins
69
Are all zinc-finger proteins involved in DNA binding?
- no, some in RNA and protein interactions
70
What is the structure of zinc-finger proteins?
- β, β, α protein fold centred around central Zn2+
71
How do zinc-finger protein perform their role?
- helix slots into major groove and forms specific interactions w/ adj nucleotides
- zinc-finger proteins can be designed to target specific seqs
- Zn2+ has contacts w/ 4 AAs (2 Cys and 2 His or 4 Cys)
- -> found as 2 closely spaced pairs of residues in 1° seq, separated by intervening seq thats looped when drawn out (= zinc 'finger')
- Zn2+ stabilises 3D arrangement of α helix and β sheets
72
What are the 2 major families of zinc finger proteins?
- C2H2 = contain multiple zinc fingers and bind DNA as monomers
- C4 = in nuclear receptors, contain 2 zinc fingers and bind as dimers
73
What are basic leucine zipper proteins?
- family that comprise of Leu zipper to allow dimerisation
74
What are the features of basic leucine zipper proteins that allow them to perform their role?
- coiled-coil dimer linked through parallel amphipathic α helices
- w/ Leu at every 7th position --> contributes strongly to hydrophobic interactions between 2 α helices
- stably bound to by add ionic interactions
- region rich in basic residues to allow DNA binding
- extended helices grip DNA of adj major grooves
- basic helix-loop-helix proteins structurally related to bZIP proteins but have non helical regions that connect Leu zipper region and helix involved in DNA binding
75
How do TF interactions diversify transcriptional control?
- TFs typically function as homo or heterodimers
- some heterodimers recognise same DNA seqs --> so target genes respond to distinct combos of activation domains under diff conditions
- combo of diff TFs can gen alt heterodimers --> increase diversity of DNA seqs, so increase pot targets
- bZIP proteins can also bind repressors, blocking DNA binding
76
How can combinatorial control also be achieved?
- w/ sTFs that composite DNA seq elements
77
How does cooperative binding of distinct DNA binding proteins occur?
- 2 proteins that'd bind weakly to adj sites w/in DNA in isolation may be able to form stable transcrip complex through intermolecular protein-protein interactions
- dep upon relative position of 2 recognition elements w/in DNA
- multiple weak interactions often underlie specificity of transcrip control
78
How is euk DNA packaged into chromatin?
- DNA of interphase packaged together w/ histones, non histone proteins and RNA into chromatin
- further compaction req to make metaphase chromosomes
79
Why must DNA be packaged?
- to fit into nucleus
- but still remain accessible at mol level for transcrip, rep and repair
- prevents other factors from binding
80
How much of nucleus is made up of DNA?
- ≈ 5% vol
| - ≈ 60% vol of sperm cell
81
What are chromosome territories?
- indiv chromosomes occupy own specific area of euk cell nucleus
82
What happens to chromosome territories during transcrip?
- decondensed
- actively transcribed genes found in DNA that's looped out
- multiple actively transcribed genes assoc w/ RNA pol and factors in transcri factories typically close to nuclear pores
83
Why does chromatin extracted from nuclei under low salt conditions resemble 'beads on a string'?
- particles are nucleosomes
- joined together by linker DNA
- nucleosomes consist of DNA wound around 1 of histone proteins
84
How is chromatin organised?
- chromatin extracted from nuclei under low salt conditions resembles 'beads on a string'
- when extracted under physiological salt conditions, more condensed form, ≈ 30nm fibre
- further packaged into higher order structure
85
What is the structure of nucleosome particle?
- octameric core, w/ 2 copies of H2A, H2B, H3 and H4
- 2 H2A/H2B and H3/H4 dimers interleaved in handshake like interaction
- 147bp of DNA makes 1.7 left handed turns round core
- length of linker DNA varies between 10 and ≈ 100bp
- H1 binds DNA as it enters and exits nucleosomes
86
When are nucleosomes released?
- limited nuclease digestion of chromatin
87
What is the structure of histones?
- core histones one of most conserved euk proteins
- histone variants found in some nucleosomes
- consist of globular protein domain w/ flexible extensions at N-ter or both ter
88
How is most DNA w/in sperm packaged?
- w/ other Arg ich proteins called protamines
89
What is the structure of 30nm fibre of chromatin?
- in vitro, 10nm fibre condenses into "30nm fibre" at physiological salt conditions
- packaging req histone tails and linker histone H1
- 2 classes of model proposed for structural organisation
- -> single helix
- -> 2 stranded left-handed double helix of nucleosomes
90
How can core histone tails be post translationally mod?
- acetylation of Lys
- mono/dimerisation of Lys
- mono/symmetrically dimethylation/asymmetrically dimethylation of Arg
- phosphorylation of Ser/Thr
- ubiquitylation of Lys
91
Are post translational mods of core histone tails mutually exclusive or dep?
- can be either
- mutually exclusive = eg. methylation of Lys blocks acetylation
- mutually dep = eg. ubiquitination of H2B req for methylation of H3K4
92
How is the Lys side chain of histone mod?
- most involve ε-amino group of Lys
- histone acetyltransferases add acetyl groups to neutralise +ve charge
- readily reversed by histone deacetylase complexes
- methyl groups slowly removed by lysine-specific demethylases (allowing deacetylation to occur)
93
How are heterochromatin and euchromatin packaged in interphase cells?
- heterochromatin = densely packaged
| - euchromatin = loosely packaged
94
What does constitutive heterochromatin contain, and how is it inherited?
- repetitive DNA seqs = telomeres, centromeres and satellite DNA
- stably inherited through cell division
95
What is the structure of facultative heterochromatin?
- densely packaged but able to decondense
96
What is heterochromatin now used to refer to?
- transcriptionally inactive DNA
97
What are the 2 markers of heterochromatin?
- MeH3K9 (meth H3 at position Lys 9)
| - MeH3K27
98
What are the 2 markers of actively transcribed DNA?
- AcH3K9
| - MeH3K4
99
What is methylation and acetylation of core histones important for?
- structural transition of chromatin between condensed and decondensed state
100
Where are diffs between mod patterns of core histones found between?
- actively transcribed genes and heterochromatin
101
What is the histone code?
- histone mods affect chromatin structure both in trans (bound by factors) and in cis (nucleosome interactions)
102
What is the purpose of ChIP (chromatin immunoprecipitation)?
- shows histone mod status of genes
| - allows analysis of protein/DNA interactions in vivo
103
How is ChIP carried out?
- interactions stabilised by chemical crosslinking (covalently links DNA to protein)
- DNA fragmented and protein/DNA complexes purified by immunoprecipitation (using diff antibodies)
- DNA analysed by PCR or microarray
- antibodies specific to acetylated or methylated histones and gene chips provide genome wide histone maps
104
How can chromodomain bind methylated histone tails?
- reading histone code req proteins that specifically recognise mod nucleosomes
methylated H3K9 mark req for heterochromatin formation --> recognised by proteins containing chromodomain
- heterochromatin protein I (HPI) is most abundant protein in heterochromatin --> H3 peptide binds HPI as 'missing' strand in structure
- many chromodomain proteins also have adj structurally related chromoshadow domain
--> allows interaction w/ other chromoshadow domain proteins
- ∴ HPI can draw together adj nucleosomes carrying H3M9me3 mark
105
How is heterochromatin formed by HPI and histone methyltransferases (HMTs)?
- heterochromatin spreads from initiation point and extends until boundary elements (spreading req nucleosomes to be present)
- spreading dep upon H3K9 trimethylation and req HP-I and Suv3-9
- Suv3-9 has chromodomain --> HMT activity stimulated by interaction of chromodomain to adj H3K9me3 nucleosomes
106
How does chromatin-mediated repression occur in yeast?
- heterochromatin silences gene expression at rDNA locus, telomeres, centromeres and mating type locus
- gene conversion occurs at active MAT locus upon cell division
- repression of HMLα and HMRa loci is dep upon adj silencer regions
- heterochromatin formation promoted through histone deacetylase activity
- protein RAP1 binds DNA in silencer regions, then recruits Sir proteins
- Sir2 removes acetyl groups from histones, then can bind to deacetylated histones and adj nucleosome deacetlyated
- histone hypoacetylation allows chromatin condensation
107
What is the role of chromatin boundary elements?
- define edges of heterochromatic regions
108
What is the role and structure of insulator elements?
- prevent heterochromatin spreading into actively transcribed genes
- block cross activation or repression enhancer/silencer elements
- consist of clusters of binding sites for specific DNA binding proteins
109
Where is the nuclear matrix found, and what is its role?
- lies outside chromatin regions of nucleus
| - structural framework fo rmol processes
110
What is the role of matric attachment regions w/in DNA?
- tether chromatin to nuclear matrix and demarcate chromatin domains
111
What are polycomb protein complexes and trithorax protein complexes critical for?
- polycomb = maintenance of heterochromatin of Hox genes
| - trithorax = maintain Hox genes in transcriptionally active site
112
What do polycomb protein complexes contain?
- H3K27-specific histone methyltransferase
| - enhancer of zest
113
What do trithorax protein complexes contain?
- contain histone methyltransferase that introd H3K4 epigenetic mark for transcriptionally active chromatin
114
What domain do many histone methyltransferases contain?
- catalytic SET domain
115
What are hypersensitive sites (in DNA)?
- rapidly degraded upon incubation w/ DNA
116
How was it shown that nuclease-hypersensitive sites are in actively transcribed genes?
- digestion of chromatin w/ micrococcal nuclease or DNase I can release nucleosomes
- -> DNA assoc w/ nucleosomes protected, whereas nucleosome free DNA is nuclease snesitive
- -> resolved through agarose gels
- DNA hybridised to gene specific probes
- showed that 5' end of globin gene nuclease sensitive in cells where its actively transcribed
- S blotting to see if particular region of DNA in nucleosome
117
What do nucleosome free regions in promoters and terminators allow to be mapped?
- genome wide map of nucleosome occupancy, using histone specific antibodies
118
What did ChIP assays reveal about nucleosome free regions?
- found in promoter and terminator regions
119
Nucleosomes adj to nucleosome free regions are enriched with what?
- euchromatin markers
120
How do activators direct histone acetylation of promoters?
- histone deacetyltransferase complex SAGA is a coactivator
- recruited to UAS seqs through interaction w/ transcriptional coactivators, eg. Gcn4 and Gal4
- acetylation of histones loosens nucleosome interactions and provides recognition sites for proteins w/ bromodomains
- -> eg. chromatin remodelling machines and the general TF, TFIID
121
What is the catalytic component of SAGA complex, and what is its role? (not on slides)
- Gcn5
- triggers acetylation of multiple sites w/in core histones
- hyperacetylation leads to decondensation of nucleosome structure at level of nucleosome interactions and by recruiting other factors that promote open chromatin structure
- proteins w/ bromodomains then able to interact w/ acetylated histones
122
What is the role of chromatin remodelling complexes?
- have ATP dep DNA helicase activity that can push DNA into nucleosomes, causing them to "slide" along DNA
- bind to activation/repression domains of TFs
123
How do repressors direct histone deacetylation of promoters?
- Sin3/pd3 histone deacetylase complex is transcriptional repressor (Rpd3 is catalytic subunit)
- recruited to upstream regulatory seqs of many genes through interaction w/ repression domain of Ume6 by Sin3
- deacetylation of core histones leads to chromatin condensation and subsequent transcriptional repression
- other corepressor complexes contain histone methyltransferases (in mammalian cells)
124
How does DNA methylation cause transcriptional activation?
- cells in higher euks express cytosine methyltransferases
- DNA methylation of CpG islands correlates w/ transcriptional repression
- m5c sites recognised by proteins such as MeCP2
- binding of MeCP2 recruits HDAC (histone deacetylase) or HMT (histone methyltransferase) complexes
125
What is position effect variegation?
- repositioning normally well expressed gene close to heterochromatic gene can potentially result in its transcriptional repression
126
Are genes next to heterochromatin expressed?
- can be in some cells, but not others
| - prod mosaic expression pathway
127
What are some examples of position effect variegation?
- mottled allele of white eye locus in fruit flies
| - telomere position effect in budding yeast
128
What is X chromosome inactivation, and how is it controlled?
- random inactivation of maternal/paternal X chromosome to allow dosage compensation
- silenced chromosome visible as Barr body
- controlled by X inactivation centre
--> encodes no. noncoding RNAs, inc Xist transcript
- Xist retained in nucleus and binds along length of inactive X chromosome
-
129
What is a Barr body?
- how inactivated chromosome visualised
130
What is Xist, and what is its role?
- X inactive specific transcript
- retained in nucleus and binds along length of inactive X chromosome
- recruits polycomb repressor complex 2, which contains Enhancer of zest and induces heterochromatin formation
- expression reg by other noncoding RNAs encoded in X inactivation centre, inc Tsix (its antisense transcript
131
How does RNA mediated trancsrip repression occur in yeast?
- H3K4me found downstream of Gal10 gene during growth in glucose
- Gal10 noncoding RNA silences GAL expression by recruiting HDACs and HMT, leading to chromatin condensation
- heterochromatin stably inherited, so only low levels of such noncoding RNAs req for efficient suppression
132
What are unstable RNAs gen in transcrip called in yeast?
- cryptic unstable transcripts
133
Where are unstable RNAs gen from in euks?
- nucleosome free regions in promoters of normally expressed genes
134
How do euk promoters gen unstable RNAs?
- transcrip from some promoters can be bidirectional, gen either protein coding transcript or unstable noncoding RNA
- -> true of many pol II promoters in euk cells
- -> can "toggle" in single cell, allowing rapid response to regulatory signals
135
How can noncoding RNAs block TF binding?
- SER3 gene in yeast req for Ser biosynthesis and tightly repressed in rich medium
- intergenic region upstream of SER3 actively transcribed, gen noncoding RNA, SRG1
- active transcript of SRG1 blocks binding of TFs at SER3 promoter by transcription interference mechanism
136
What is RNA interference used for?
- deplete specific gene products and underlies natural mechanism of gene reg
137
How is RNA interference carried out?
- dsRNA digested into ≈25nt fragments by endonuclease Dicer (makes staggered cuts)
- 1 strand incorp into RNA-induced silencing complexes or RNA-induced transcriptional silencing complexes
- complexes targeted through bping to specific mRNAs or genes, leading to mRNA degradation or transcriptional repression
138
How can RNA interference be used on centromeric heterochromatin in fission yeast?
- upon mitosis, phosphorylation of H3S10 causes HPI depletion
- DNA rep dilutes histone heterochromatin marks and allows brief bidirectional transcrip pericentromeric repeats
- RNA interference machinery gen RITS complex that recruit Suv3-9 to centromere and restore HPI binding and heterochromatin formation
139
What factors of sTFs are key to expression of a given gene?
- effective conc
- availability
- activity
140
By what pathways can TF activity be regulated?
- de novo synthesis = prod more of TF
- ligand binding = prod active TF once bound to ligand
- post translational mod = eg. phosphorylation
- formation of protein complex = most proteins don't function as monomers, many do at heterodimers
- release of inhibitor molecule = disassemble protein/inhibitor complex to release active protein
- proteolytic activation = activated through proteolytic cleavage
141
How is TF activity reg?
- often in response to extracellular signal
- signalling molecules recognised by specific transmembrane cell surface receptors
- -> signalling molecule doesn't enter cell
- -> induces response in receptor, triggering signal to be relayed w/in cell via other molecules
- -> sTF activated
- alt, small lipid soluble hormones pass through plasma membrane and bind directly to TF
142
What are intracellular receptors which are also TFs?
- nuclear receptors
143
What is an orphan receptor?
- receptor without identified ligand
144
What some examples of common lipid soluble hormones, and what is their structure
- cortisol, retinol, thyroxine
- heterozytic ring structures (1 or 2 rings)
- lipid soluble so can pass through membrane
- soluble in cytosol
- bind specifically to TFs (then TFs = nuclear receptors)
145
What is the domain organisation of nuclear receptors?
- highly conserved central C4 zinc finger binding domain
- C-ter ligand binding domain
- N-ter activation/repression domain --> diff variants arise through alt splicing and phosphorylation, and responsible for differential response of receptors in diff cell types
- typically function as homomeric or heterodimeric dimers
146
What do DNA binding domains have nuclear receptors contain, and what is their role?
- 2 C4 zinc finger domains
- proximal zinc finger recognises response element
- distal zinc finger mediates dimerisation
147
What is role of homomeric vs heterodimeric nuclear receptors?
- homomeric (eg. estrogen receptor) bind palindromic repeats
- heteromeric (eg. retinoic acid receptor) bind direct or inverted repeat seqs and contain common monomer, retinoid X monomer
148
How are heterodimeric nuclear receptors activated to promote transcrip initiation?
- localised only in nucleus
- in absence of ligand RXR bound t corepressor
- -> recruits HDACs and blocks transcrip
- ligand bound to nuclear receptor displaces corepressor protein from RXR
- heterodimeric receptor binds HAT complexes that hyperacetylate nucleosomes and interacts w/ mediator complexes
149
How are homodimeric nuclear receptors activated to activate transcrip?
- localised in cytp in absence of ligand
- anchored in complexes w/ inhibitor protein = heat shock proteins
- hormone binds receptor when enters cell
- -> releases nuclear receptor when enters cell
- -> ligand binding domain of homodimeric nuclear receptor contains nuclear localisation signal and is sufficient for ligand dep import into nucleus
150
What is the general cell surface receptor signalling pathway?
- binding of ligand to cell surface receptors triggers formation of receptor complex (typically dimerisation of receptor)
- transduction involves activation of 1 or more protein kinases
- receptor can directly activate TF or multiple steps
- sometimes 2° signalling molecule gen w/in cytosol
- TFs activated in nucleus or cyto
151
Are receptor molecules specific?
- no, can induce more than 1 response pathway
152
What are cytokines, and what are they important for?
- small secreted polypeptides
| - important for cell growth and differentiation
153
What are JAKs?
- protein kinases assoc w/ cytokine receptors
154
What happens during JAK/STAT pathway?
- erythropoietin prod by kidney in response to decrease blood oxygen
- induces RBC proliferation by stimulating expression of anti-apoptotic factor Bcl-xL by binding STAT5
- dimerisation causes JAK2 kinases to be brought closer to phosphorylate each other
- decreases KM, so increased kinase activity
- SH2 STAT domain activated by JAK2
- STAT dissociates and dimerises
- exposing nuclear localisation signal, enabling import into nucleus
155
What are STAT proteins, and what is their structure?
- signal transduction and activation of transcrip
| - SH2 domain, w/ ATP binding affinity
156
What activity do receptor tyrosine kinases have and how is it activated?
- intrinsic kinase activity
- activated by protein hormones and cell type specific growth factors
- interaction w/ ligand triggers receptor dimerisation, dimerised receptor autophosphorylates, activating kinase activity
157
What receptor tyrosine kinases do human cells express for epidermal GF?
- HER1, HER2, HER3, HER4
158
How were many receptor tyrosine kinases initially identified?
- cancer studies
159
How is HER2 overexpression important in many breast cancers?
- HER2 doesn't directly bind ligand
- forms heterodimeric complexes w/ HER1, 2 and 4
- overexpression makes cells responsive to low levels of GF = constitutive growth
- HER2 amplification in 25% breast cancers
160
What is Ras, and what is its role?
- small membrane bound GTPase molecular switch
| - triggers kinase cascade ending in MAP kinases that enter nucleus and target sTFs
161
When is Ras active/inactive?
- active when bound to GTP, by GEFs
| - inactive when bound to GDP, by GAPs
162
Where are mutations w/ RTK/Ras/MAP kinase pathway found?
- wide range of tumours
163
How is the TF, CREB activated?
- through cAMP/protein kinase A pathway
- induced by GPCRs
- binding of ligand enables receptor to function as GEF --> activates membrane anchored adenylyl cyclase via trimeric G protein
- adenylyl cyclase converts ATP --> cAMP
- cAMP causes release of catalytic subunit of protein kinase A
- translocates into nucleus and activates CREB
- phosphorylated CREB binds CREs in target genes and binds to coactivator CBP
164
What are the diverse functions of RNA binding proteins?
- mediate specific steps in transcrip, RNA processing, mRNA export, localisation , degradation and translation
165
Do RNA binding proteins bind specifically or nonspecifically?
- some bind specific seqs at low nM Kd values
| - others bind nonspecifically
166
How can genome wide analyses of RNA binding sites be performed at nt resolution?
- iCLIP and CRAC techniques
- assoc RNA and protein covalently crosslinked in vivo by UV irradiation
- RBP purified using specific antibody or purification of epitope-tagged fusion protein
- bound RNA degraded to short fragment, gen cDNA and seq using high throughput seqs
- cross linked nts misread by reverse transcriptase and appear as seq error
- identifying substrates of processing enzymes, so not limited to analysing stable complexes
167
What are some characteristics of RNA binding proteins?
- highly abundant
- most ubiquitously expressed
- half assoc w/ mRNA
- NA binding domains have common ancestories, TFs have more seq homology
- many RNA binding domains in multiple repeats or combos
168
How can basic rich seqs mod RNA?
- eg. RGG box
- often regulate but not sufficient for stable binding
- multiple low affinity reactions can cumulatively contribute to specific binding
169
What is the structure of RNA recognition motif?
- β sheet supported by 2 α helices
- consensus seq w/in 2 central β-strands
- β sheet 1° interaction for RNA recognition
- contacts w/in loops between β-strands can confer specificity
170
What is the role of RNA recognition motifs?
- v high affinity for RNA
- often 2 or more function together
- many also medite protein interactions
171
What does U1A bind to?
- U1 snRNA w/ high affinity
172
How does poly(A) binding protein bind poly(A) RNA?
- contains 4 distinct RNA recognition motifs
- 2 RRMs req
- β sheets make extended binding surface
- poly(A) chain along length of surface
- can interact w/ 7 adenosine res (each distinctly recognised)
- interaction characterised by extensive aromatic base and base-base stacking interactions
173
Where is KH domain found?
- K homology domains found in multiple copies and bind RNA or ssDNA
174
What is KH domain?
- ancient RNA binding domain
175
What is structure of KH domain?
- 2 variant forms, both w/ βααβ core
- 2 α helices connected by loop w/ highly conserved GXXG seq and variable loop
- nts splayed out onto hydrophobic cleft formed
- no stacking interactions between aromatic abses and nts
176
What are Sm and Lsm complexes, and what is their structure?
- small proteins w/ αβ1-5 structure which forms bent 5 stranded β sheet
- 7 proteins assemble into ring structure
- U-rich RNA seq binds along inner ring surface
- each nt stacked between aromatic and basic residues
- WC functional groups recognised by specific H bonding interactions w/ Asn
177
How does modular recognition of RNA by puf domains occur?
- pumilio homology domains contain 8 consecutive Puf repeats
- Puf repeats stacked giving curved structure
- consensus aromatic, basic and acidic residues line concaved surface
- PUM-HD binds 10nts, 8 specifically recognised by 3 AA residues in each repeat
- few contacts between nts
- bases stacked between side chains of residues
- specific base recognition involves H bonding w/ key AAs
178
Which proteins is ds RNA binding domain found in?
- diverse range
- inc RNase III, protein kinase R, RNA-dep adenosine deaminase
- euk proteins can contain multiple
179
What is role of ds RNA binding domain?
- 65 res domain interacts w/ dsRNA in seq-indep manner, through α helices stabilised by β sheet
- RNase III and related enzymes function in processing rRNA, tRNA and microRNAs
180
What is the mechanism of splicing?
- 2 transesterification reactions (so no ATP req)
| - intron lariat degraded by debranching enzyme and RNases
181
What is the spliceosome, and what is its role?
- large dynamic complex of 5 distinct small nuclear ribonucleoproteins (snurps = U1, 2, 4, 5, 6) and other proteins
- carries out nuclear pre-mRNA splicing
- assembled and disassembled on pre-mRNA during each splicing event
182
What do snurps contain?
- 1 snRNA and 6-10 proteins
- Sm proteins common to all pol II snurps
- U6 bound to Lsm proteins
183
What do snurps do?
- bp w/ pre-mRNA and w/ each other during splicing
184
How is spliceosome assembled on pre-mRNA?
- bping between snRNAs and pre-mRNA, or between indiv snRNAs is key
- U1 snRNP binds 5' splice site
- U2 snRNP binds branchpoint A
- U4/U5/U6 assoc together as tri-snurp and bind to U1 and U2 snurps
- U1, then U4 released
- U5/U6/U2 remain in catalytic spliceosome and U6/U2 snurps catalyse splicing
185
What is the spliceosome cycle?
- spliceosomes undergo cycles of assembly, catalysis and disassembly
- each step assoc w/ structural rearrangements of spliceosome complex
- dep upon ATP hydrolysis
186
Why does spliceosome cycle req ATP?
- structural rearrangements req ATP to bring catalytic active form of spliceosome together
- but chem reactions themselves don't
187
What is the role of RNA helicases in pre-mRNA splicing
- ATP hydrolysis to function as chaperones to drive structural transitions
- some function as pre-mRNA fidelity factors
- ATP hydrolysis rates coupled to substrate discard pathways = kinetic proofreading
- ATPase Prp28 allows assembly of U4/U5/U6 tri-snurp onto pre-mRNA
- Snu114 and Brr2 induce U4/U6 unwinding
- Prp2 promotes formation of active spliceosome
- Prp16 and Prp22 induce conversion of complex C to post splicing complex and removal of spliced intron
- other helicases promote spliceosome recycling
188
How does kinetic proofreading in pre-mRNA splicing occur?
- Prp16 facilitates transition between 1st and 2nd step of splicing
- prp16 mutants w/ decreased ATP hydrolysis rates improve splicing of suboptimal substrates
- if exons not correctly aligned after ATP hydrolysis, pre-mRNA released and degraded
- slowed ATP hydrolysis rate enables further time for exon alignment and productive splicing
189
How can mRNA splicing be analysed in vivo?
- use 32P labelled RNA substrates and nuclear extracts
- RNAs resolved by PAGE
- looped RNA migrates slower than linear
- substrates w/ alt seq or extracts depleted of specific proteins can be assayed
- can determine if particular protein involved in splicing or if particular nt essential for splicing function
190
What is the role of C-ter domains in RNA pol II?
- C-ter domain of RNA pol II comprises multiple copies of Ser-rich heptapeptide repeat
- C-ter domain differentially phosphorylated during transcrip
- diff phosphorylation patterns of C-ter domain code allow recruitment of diff processing complexes at beginning (capping complex), middle (spliceosome) and end (cleavage/polyadenylation) of transcrip
191
How are exon boundaries defined?
- through protein interactions that bridge U1 bound at 5' end of intron w/ U2 bound at 3' end
192
What protein-RNA and protein-protein interactions occur when splicing factors promote exon/intron recognition?
- U2AF --> aids U2 snRNP binding to branchpoint
- exonic splicing enhancers
- SR proteins --> recognise ESEs and interact though RNA recognition motifs
193
How can some exceptionally large introns be spliced?
- recursive splicing mechanism
| - intron removed piecemeal by regen functional 5' splice site at spliced junction
194
Why is it so important that splicing is accurate?
- 1/3 human genetic diseases thought to be die to defect in pre-mRNA splicing
195
What is an eg of a disease caused by inaccuracies in pre-mRNA splicing?
- spinal muscular atrophy
- in exon 7 of SMN gene
- point mutation in exonic splicing enhancer blocks binding of SF2 and use of adjacent 3’ splice site
196
What are alt splicing patterns?
- each pre-mRNA can gen more than 1 coding seq
- increasing diversity of coding pot
- can occur in tissue specific manner, or w'in same cell as result of programmed change
- choice of using strong canonical site or weak cryptic splice site, decided by availability of alt splicing actors (can activate or repress spicing at any given site)
- alt splicing factors are RBPs that recognise pre-mRNA seqs knows as enhancers or silencers
197
How is sex determined in Drosophila?
- sex lethal (sxl) is splice site repressor that promotes own expression and that of transformer (tra) in female flies by exon exclusion
- in early embryogenesis sxl transcribed from PE promoter in females
- in late embryogenesis sxl trancrived from PL promoter in males and females
- tra w/ tra2 (SR protein) activates splicing at alt 3' splicing site in doublesex transcript
- isoforms of dsx act as transcrip repressors for genes req for sexual differentiation in opp sex
198
What is the catalytic mechanism of self splicing introns?
- self splicing introns are autocatalytic ribozymes
- group II introns splice using analogous mechanism w/ branchpoint adenosine and gen lariat intermediate
- group I introns dep on guanosine cofactor and gen lariat intermediate
199
What is the importance of group I introns having specific 3D structure?
- restricted to seqs that can fold into approp structures to allow juxtaposition of splice sites and guanosine nnt
- snRNPs mediate positioning of pre-mRNA splice sites
- splicing apparatus facilitated evo of intorn seqs
200
What is the role of nuclear pore complex?
- transport of protein and RNA across nuclear membrane
201
What is the structure of nuclear pore complexes?
- large but contain only ≈30 diff nucleoporin proteins
- channel of nuclear pore lined w/ EG nucleoporins (restricts passive diffusion)
- -> larger proteins and complexes transported across pore in folded state by fd
202
How is an in vivo nuclear protein import assay carried out?
- import of fluorescently labelled reporter proteins into nucleus dep upon cytoplasmic cell extract
- req presence of nuclear localisation signal
- assaying fractionated cytosolic extracts identified key transport factors, eg. Ran
203
What is Ran, and where is it found?
- small GTPase
- Ran/GEF nuclear
- Ran/GAP is cytoplasmic
204
How is Ran/GTP cycle coupled to transport?
- nuclear portein transport mediated by specific carrier proteins called karyopherins
- karyopherins can diffuse through nuclear pores and interact w/ hydrophobic FG repeat nucleoporins
- bind tightly to Ran/GTP but only weakly to Ran/GDP
205
What is the overall mechanism for nuclear import of proteins?
- importins bind to their cargo proteins via NLS seqs
| - binding of importin/cargo complex to Ran/GTP in nucleus causes release of cargo proteins
206
What is the role of exportins?
- transport proteins out of nucleus
- bind cargo via nuclear export signals
- bind cargo only when assoc w/ Ran/GTP
- bound effectively in nucleus and released upon Ran/exportin dissociation in cyto
207
How is the directionality of transport ensured?
- differential localisation of Ran/GEF and Ran/GAP
- affinity of karyopherins for their cargo and for GTP-bound Ran
- differential affinity of karyopherins for their cargos upon interaction/loss of Ran binding
208
How is mRNA export mediated by TAP?
- mRNAs exported in Ran-indep manner that uses distinct transporter protein (=TAP)
- TAP binds RNA and FG repeat nucleoporins
- TAP depletion blocks mRNA export
- mRNA binding of TAP dep upon Ref
- binding of Ref to TAP destabilises interaction w/ RNA
- RNA dissocites from Ref and binds TAP
- only spliced mRNPs exported to cyto
- TAP/mRNP complex disassembled by RNA helicase Dbp5, during mRNP remodelling
209
What is Ref?
- component of exon junction complex that is deposited on mRNA during splicing
210
How can mRNA be localised to specific regions of cell?
- random diffusion and anchoring
- active transport
- selective degradation
211
What is a zip code binding protein?
- protein which binds zip codes (= seq elements w/in mRNAs that mediate localisation )
212
Where are zip code seqs?
- 3' untranslated region
213
How is ASH1 mRNA localised in daughter cells?
- HO endonuclease induces mating type switching in S. cerevisiae, in each round of cell division
- switching only in mother cell as HO transcrip repressed in daughter cell
- ASH1 localises to daughter cell during cell division
- ASH1 mRNA localisation dep on zip code binding protein She2, She3 and Myo4
214
What is the rate limiting step of translation?
- initiation
215
How is translational controlled globally?
- mechanisms typically impact on activity of translational apparatus
- translational efficiency of indiv mRNAs med by specific RBPs and typically involves impeding translation of that mRNA
216
How is translation controlled globally by eIF2α phosphorylation?
- translation globally downreg in response to cellular stress
- stress activated protein kinases block translation by phosphorylation of α subunit of eIF2
- phosphorylated eIF2 functions as comp inhibiot of eIF2 by binding its GEF, eIF2B
- mRNAs encoding some stress induced proteins more efficiently translated at lower eIF2 levels
217
How do eIF4E/eIF4G/PABP interactions stimulate translation?
- mRNA caps and poly(A) tails recognised and bound by cap-binding complex (eIF4E/eIF4A/eIF4G) and poly(A) BP, respectively
- eIF4G/PABP interaction circularises mRNA and stimulates translation
- 4EBPs disrupt eIF4E/eIF4G interaction and block translation
218
How is translation of 5' TOP mRNAs reg by mTOR?
- 4EBPs are major target s of protein kinase mTOR
- mTOR inhibited by rapamycin
- target mRNAs contain 5' terminal oligopyrimidine motif
- mTOR pathway stimulates translation of 5' TOP mRNAs by phosphorylating 4EBPs, inactivating them
219
What is the role of mTOR?
- involved in several signalling pathways that promote cell growth and proliferation
- 1 target of mTOR signalling is translational apparatus
220
How is translation controlled by makin maternal mRNAs?
- mRNAs w'in oocytes maintained in translationally inactive form in cyto and activated by cytoplasmic polyadenylation
- translational reg of masked mRNP important in early metazoan oocyte dev
- in inactive site, maskin blocks binding between eIF4E and eIF4G
- stored mRNPs contain cytoplasmic polyadenylation element, w/in 3' untranslated regions, bound by CPE BP, and maskin
- upon fertilisation, phosphorylation of CPE BP causes dissociation of maskin and deadenylase, allowing extension of poly(A) tail and circularisation of mRNP particle
221
How is translation controlled by ribosome recruitment?
- many ribosomal proteins in E. COli autoreg own expression
- mRNA and rRNA binding sites have similar structures
- binding to mRNA blocks ribosome binding
- expression of mammalian ferritin reg by Fe regulatory proteins
- Fe response element binds to 5' leader and blocks binding of 43S preinitiation complex to cap binding complex
222
How does sxl repression translation of msl-2 occur?
- male sex lethal 2 (msl-2) expressed in male flies and is component of dosage compensation complex
- sxl protein binds to 3' untranslated region of msl-2 and blocks interaction of 43S preinitiation complex w/ cap binding complex
- for transcripts that bypass this reg, sxl also functions as failsafe mechanism --> binds to 5' leader seq of msl-2 mRNA and blocks scanning ribosome
223
Why do mRNA poly(A) tails decrease in length over time?
- slowly shortened in cyto by deadenylases
| - deadenylated mRNAs raipdly degraded
224
What is the lifetime of mammalian mRNAs?
- varies from less than 10 mins to oveer 9 hours
225
How are mRNA stability and translational efficiency related?
- inversely correlated
226
What is the euk mRNA turnover pathway?
- after deadenylation, m7G cap structure removed by action of heterodimeric capping enzyme
- alt, deadenylated transcripts can be degraded by 3' --> 5' exoribonucleases
- deadenylation and decapping are rate-limiting steps in mRNA decay
227
How does mRNA stability affect expression level?
- expression level of mRNA reflects rate of prod and turnover
- so stable mRNAs expressed at higher levels than unstable if under same transcrip control
- upon transcrip induction/repression, unstable mRNA achieves new steady state more rapidly than stable
- changes in mRNA stability cause rapid change in steady state levels in transcrip rate remains constant
228
How is mRNA turnover reg by A/U rich element (ARE)-mediated decay
- unstable mRNAs often contains AREs
- typical AREs contain multiple AUUUA motifs w/in U rich region
- ARE mediated decay reg +vely and -vely by relative expression of ARE BPs (AUF1 and HuR respectively)
- AUF1 and HuR reg by alt splicing and post translational mod
229
How are mRNAs w/ early stop codons rapidly degraded?
- by nonsense mediated decay pathway
- dep upon mRNA being actively transcribed
- 3' UTR region of mRNAs assoc w/ proteins
- -> this 3' mRNP sensed by ribosome at termination codon
- NMD triggered in absence of interactions between ribosome and mRNP 3' domain
230
Why are truncated proteins not commonly expressed in vivo?
- most wont fold into stable structure
- degraded by proteasome complex
- euk cells have quality control systems to eradicate mRNAs that contain early stop codons
231
What is the general pathway for expression of a euk gene?
- reg of expression pathway allows change in amount of product prod
- reg occurs at rate limiting steps in pathway
- most genes reg primarily at level of transcrip
- post transcriptional reg can occur through alt splicing, localisation, translational control or mRNA degradation
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